The present invention is directed to the analysis of RNA molecules by liquid chromatography. More specifically, the invention is directed to a liquid chromatography system and method, such as Matched Ion Polynucleotide Chromatography, which enhances the purification of RNA.
RNA molecules are polymers comprising sub-units called ribonucleotides. The four ribonucleotides found in RNA comprise a common cyclic sugar, ribose, which is covalently bonded to any of the four bases, adenine (a purine), guanine (a purine), cytosine (a pyrimidine), and uracil (a pyrimidine), referred to herein as A, G, C, and U respectively. A variety of modified bases are also encountered in RNA. A phosphate group links a 3xe2x80x2-hydroxyl of one ribonucleotide with the 5xe2x80x2-hydroxyl of another ribonucleotide to form a polymeric chain. Secondary structure commonly occurs via intra-chain hydrogen bonds between complementary bases.
Ribonucleic acid (RNA) transports genetic information within the cell. In the most general terms, RNA passes specific peptide and protein coding information from the genome in the nucleus to those parts of the cell responsible for the production of these peptides and proteins. There are a number of different RNAs found in the cell at any given time. Some of them are noted below, along with their functions:
Messenger RNA (mRNA): Carries the coding messages to the ribosomes for the production of peptides and proteins;
Small nuclear RNA (snRNA): Responsible for removing intronic sequences from mRNA precursors (preribosomal mRNAs), prior to the spliced mRNA delivery to the ribosomes;
Transfer RNA (tRNA): Provide chemically activated amino acids for binding to the ribosomal complex in the process of protein synthesis;
Ribosomal RNA (rRNA): RNA within the ribosomes themselves, which by association, are part of the protein synthesis process.
Within molecular biology, it is often necessary to isolate these RNA molecules, particularly mRNA. This is because an mRNA xe2x80x9cmessagexe2x80x9d indicates that a gene has been transcribed. Furthermore, the extent to which the gene is expressed (up-regulated, down-regulated, turned on, turned off) is often proportional to the amount of gene-specific RNA (mRNA) present in the cell. The quantitation of gene expression via mRNA content can occur by various means (e.g., RT-PCR, expression array/hybridization analysis, etc.)
On other occasions it is desirable to create a compilation, or xe2x80x9clibraryxe2x80x9d, of those genetic messages being expressed in a cell or cells under a given set of conditions (i.e., normal vs. diseased state). This is often performed by selectively harvesting the mRNAs present in a sample, reverse transcribing the mRNAs to cDNA (first strands and second strands), and then cloning these double stranded sequences into some suitable vector. Once cloned, the cDNA xe2x80x9clibrariesxe2x80x9d can be utilized in various procedures.
RNA is thus the starting material in numerous molecular biology experiments involving the identification of unknown genes and assignment of functions to various proteins. Quality, quantity, purity, and size distribution of RNA determine the rate of success in applications such as CDNA library construction, Northern blot analysis, reverse transcription, and in situ analysis. Present techniques for the purification of RNA are labor intensive and lengthy. Quantification is routinely performed by gel visualization, radiometric methods, and spectrophotometric techniques. Sizing and quality determination is often performed by electrophoresis on denaturing agarose gels. However, RNA can become covalently modified by the chemicals used during the fractionation process (e.g., formaldehyde or acryalmide). Many of the present separation techniques require the use of hazardous chemicals (e.g., methylmercuric hydroxide).
In the preparation of mRNA from total RNA, spin columns containing beads coated with poly T oligomers are often used (e.g., Poly(A)Pure(trademark) mRNA Purification Kit, Ambion, Inc., Austin, Tex.; Oligotex(trademark) mRNA Purification System, Qiagen, Inc., Valencia, Calif.). The disadvantages of this technique include a requirement for high amounts of total RNA sample due to low recovery of mRNA, contamination of the product (e.g. by rRNA), and degradation of the mRNA product.
There is a need for faster, safer, more reliable, less labor intensive, and more accurate methods of RNA analysis.
Separations of polynucleotides such as RNA have been traditionally performed using electrophoresis through agarose gels or sedimentation through sucrose gradients. However, liquid chromatographic analysis of polynucleotides is becoming more important because of the ability to automate the process and to collect fractions.
Traditional chromatography is a separation process based on partitioning of mixture components between a xe2x80x9cstationary phasexe2x80x9d and a xe2x80x9cmobile phasexe2x80x9d. The stationary phase is provided by the surface of solid materials which can comprise many different materials in the form of particles or passageway surfaces of cellulose, silica gel, coated silica gel, polymer beads, polysaccharides, and the like. These materials can be supported on solid surfaces such as on glass plates or packed in a column. The mobile phase can be a liquid or a gas in gas chromatography.
The separation principles are generally the same regardless of the materials used, the form of the materials, or the apparatus used. The different components of a mixture have different respective degrees of solubility in the stationary phase and in the mobile phase. Therefore, as the mobile phase flows over the stationary phase, there is an equilibrium in which the sample components are partitioned between the stationary phase and the mobile phase. As the mobile phase passes through the column, the equilibrium is constantly shifted in favor of the mobile phase. This occurs because the equilibrium mixture, at any time, sees fresh mobile phase and partitions into the fresh mobile phase. As the mobile phase is carried down the column, the mobile phase sees fresh stationary phase and partitions into the stationary phase. Eventually, at the end of the column, there is no more stationary phase and the sample simply leaves the column in the mobile phase.
A separation of a mixture of components occurs because the mixture components have slightly different affinities for the stationary phase and/or solubilities in the mobile phase, and therefore have different partition equilibrium values. Therefore, the mixture components pass down the column at different rates.
In traditional liquid chromatography, a glass column is packed with stationary phase particles and mobile phase passes through the column, pulled only by gravity. However, when smaller stationary phase particles are used in the column, the pull of gravity alone is insufficient to cause the mobile phase to flow through the column. Instead, pressure must be applied. However, glass columns can only withstand about 200 psi. Passing a mobile phase through a column packed with 5 micron particles requires a pressure of about 2000 psi or more to be applied to the column. 5 to 10 micron particles are standard today. Particles smaller than 5 microns are used for especially difficult separations or certain special cases). This process is denoted by the term xe2x80x9chigh pressure liquid chromatographyxe2x80x9d or HPLC.
HPLC has enabled the use of a far greater variety of types of particles used to separate a greater variety of chemical structures than was possible with large particle gravity columns. The separation principle, however, is still the same.
An HPLC-based ion pairing chromatographic method was recently introduced to effectively separate mixtures of polynucleotides wherein the separations are based on base pair length (U.S. Pat. No. 5,585,236 to Bonn (1996); Huber, et al., Chromatographia 37:653 (1993); Huber, et al., Anal. Biochem. 212:351 (1993)). Ion pair reverse phase high pressure liquid chromatography (IPRPHPLC) was used as a process for separating DNA using non-polar separation media, wherein the process used a counterion agent, and an organic solvent to release the DNA from the separation media. This method was used in the separation of double stranded DNA of up to about 1,000 base pairs and for the separation of single stranded DNA of up to about 100 nucleotides.
Objects of the present invention include providing a method and system for segregating RNA molecules which is fast, safe, reliable, convenient, reproducible, and quantatitive.
In one aspect, the invention provides a chromatographic method for segregating a mixture of RNA molecules having lengths exceeding about 100 nucleotides. The method includes the steps of a) applying a solution of the fragments and counterion reagent to a column containing polymeric beads having non-polar surfaces, wherein said beads have an average diameter of about 1 to about 100 microns and b) eluting the RNA molecules with a mobile phase which includes the counterion reagent and an organic component. The method is preferably carried out under conditions effective to denature the secondary structure of said RNA molecules. The method can include collecting mobile phase fractions containing the segregated RNA molecules. The method can be carried out at a mobile phase temperature greater than about 50xc2x0 C., preferably greater than 60xc2x0 C., and more preferably greater than 70xc2x0 C.
In the inventive method, the mobile phase preferably includes a counterion agent and a water soluble organic solvent. The organic solvent can be selected from the group consisting of alcohol, nitrile, dimethylformamide, tetrahydrofuran, ester, ether, and mixtures of one or more thereof. A preferred solvent includes acetonitrile. The counterion agent can be selected from the group consisting of lower alkyl primary amine, lower alkyl secondary amine, lower alkyl tertiary amine, lower trialkyammonium salt, quaternary ammonium salt, and mixtures of one or more thereof. Preferably, the counterion agent is selected from the group consisting of octylammonium acetate, octadimethylammonium acetate, decylammonium acetate, octadecylammonium acetate, pyridiniumammonium acetate, cyclohexylammonium acetate, diethylammonium acetate, propylethylammonium acetate, propyldiethylammonium acetate, butylethylammonium acetate, methylhexylammonium acetate, tetramethylammonium acetate, tetraethylammonium acetate, tetrapropylammonium acetate, tetrabutylammonium acetate, dimethydiethylammonium acetate, triethylammonium acetate, tripropylammonium acetate, tributylammonium acetate, tetrapropylammonium acetate, tetrabutylammonium acetate, triethylammonium hexafluoroisopropyl alcohol, and mixtures of one or more thereof.
Also, in the inventive method, the pH of said mobile phase is preferably within the range of about pH 5 to about pH 9. A preferred pH is about pH 7.
The method can be used for analyzing RNA molecules exceeding about 1,000 nucleotides and can be used for analyzing RNA molecules having up to about 20,000 nucleotides.
In another aspect, the invention concerns a method for segregating a mixture of RNA molecules by Matched Ion Polynucleotide Chromatography, in which the mixture includes RNA molecules having lengths exceeding about 100 nucleotides. The method includes a) applying a solution of the molecules and counterion reagent to a column containing polymeric separation beads having non-polar surfaces, wherein the separation beads have an average diameter of 1 to 100 microns and the column has an ID greater than about 5 mm and b) eluting the RNA molecules with a mobile phase which includes the counterion reagent and an organic component. In the method, the eluting step preferably is carried out under conditions effective to minimize the secondary structure of the RNA molecules. The eluting can be carried out at a temperature within the range of about 40xc2x0 C. to about 80xc2x0 C., and preferably at about 75xc2x0 C. In one embodiment of the method, the column ID is greater than about 7 mm. In another embodiment, the column ID is greater than about 10 mm. In yet another embodiment, the column ID is greater than about 50 mm. In still another embodiment the column ID is in the range of about 5 mm to about 1 m.
In still another aspect, the invention concerns an improved column for segregating a mixture of RNA molecules by Matched Ion Polynucleotide Chromatography, the mixture including molecules having lengths exceeding about 100 nucleotides. The column includes a cylinder having an ID greater than about 5 mm and containing polymer beads. The beads have an average diameter of 1 to 100 microns and are unsubstituted polymer beads or are polymer beads substituted with a hydrocarbon moiety having from 1 to 1,000,000 carbons. In one embodiment, the column ID is greater than about 7 mm. In another embodiment, the column ID is greater than about 10 mm. In yet another embodiment, the column ID is greater than about 50 mm. In still another embodiment the column ID is in the range of about 5 mm to about 1 m.
In yet another aspect, the invention concerns a system for segregating a mixture or RNA molecules by Matched Ion Polynucleotide Chromatography in which the system includes a column which includes a cylinder having an ID greater than about 5 mm and containing polymer beads. The beads have an average diameter of 1 to 100 microns and are unsubstituted polymer beads or are polymer beads substituted with a hydrocarbon moiety having from 1 to 1,000,000 carbons.